CN107296955B - Immunogenic bordetella bronchiseptica compositions - Google Patents

Abstract

Provided herein are compositions, combined preparations, and methods comprising bordetella bronchiseptica and an isolated pertactin that are effective in treating or preventing respiratory infections such as kennel cough in animals.

Description

Immunogenic bordetella bronchiseptica compositions

The application is a divisional application of a patent application with the application date of 2012, 2/3, the application number of 201280015807.1 and the title of "immunogenic bordetella bronchiseptica composition".

Technical Field

The present invention relates to the field of immunology, in particular to the field of immunogenic and vaccine compositions. More particularly, the disclosure relates to compositions comprising a Bordetella bronchiseptica (Bordetella bronchiaseptica) preparation in combination with pertactin for the treatment or prevention of respiratory disease in dogs.

Background

Bordetella bronchiseptica is a gram-negative bacterium that colonizes the canine respiratory tract and causes bronchitis or "kennel cough". Hawkins, E.C., Veterinary Internal Medicine (1995), pp.767-811. Branch standBordetella tracheitis leaves dogs susceptible to and often co-existing with other respiratory agents. To date, many vaccines have been used to treat bronchitis caused by Bordetella bronchiseptica, including

CAe、

B、Univac 2、

KC2、Naramune^TM-2 and Kennel-Jec^TM 2。

However, most of the existing commercial vaccines require cumbersome intranasal administration and the addition of adjuvants, which can cause harmful side effects such as burning sensation and irritation. Viera Scheibner et al, Nexus Dec 2000(Vol 8, No 1). Intranasal vaccines are not popular among veterinary practitioners due to their own risks of being relatively irritable to the animal during vaccine administration. Subunit vaccines, such as those involving the application of the p68 protein of bordetella bronchiseptica (pertactin), have been studied but to date, may not be included in any commercial canine vaccine due to insufficient immunogenicity, reactivity and/or formulation stability.

Bordetella bronchiseptica is an important factor in canine infectious respiratory disease, a highly contagious disease common in dogs living under crowded conditions such as rescue centers and fencing or training kennels. The pathogenesis of CIRDC is considered to be multifactorial, involving several viruses and bacteria. Known infectious agents as pathogens of CIRCDC include, in addition to the bacteria Bordetella bronchiseptica (Bemis et al, Lab. anim.Sci.,29:48-52,1977), canine respiratory coronavirus (CRCoV) (Erles et al, Virology,310(2): 216-. To date, no comprehensive combination vaccine has emerged that can combat all or most of the aforementioned pathogens.

Thus, there is a need for an immunogenic composition that can be safely administered parenterally to dogs that provides long-lasting immune protection against bordetella bronchiseptica without deleterious side effects or interference with other antigens in the combination vaccine or risk to the veterinarian. The present disclosure satisfies these and other related needs.

Disclosure of Invention

In one embodiment, the invention provides an immunogenic composition comprising bordetella bronchiseptica and an isolated pertactin antigen, among others. In another embodiment, the pertactin antigen is a recombinant protein. In another embodiment, the pertactin antigen is from bordetella bronchiseptica. In another embodiment, the pertactin antigen is p 68. In another embodiment, the composition further comprises an isolated Bsp22 antigen.

In another embodiment, the bordetella bronchiseptica component is a bacterin or bacterial extract (bacterial extract). In another embodiment, the composition is non-adjuvanted. In another embodiment, the composition further comprises an adjuvant.

In another embodiment, the pertactin antigen is present at about 1 μ g to about 30 μ g. More specifically, the pertactin is present at about 5 μ g to about 20 μ g, still more specifically, about 7 μ g to about 15 μ g, even more specifically, about 5 μ g, 10 μ g, 15 μ g, or 20 μ g. Preferably, the pertactin antigen is prepared by dissolving pertactin inclusion bodies in urea and optionally purifying by column chromatography. The pertactin antigen is soluble and preferably substantially free of aggregates (aggregates).

Another embodiment provides an immunogenic composition comprising a plurality of recombinant p68 pertactin antigens, wherein the composition is substantially free of p68 aggregates. More specifically, the plurality of recombinant p68 pertactin antigens are prepared by dissolving pertactin inclusion bodies in urea and optionally purifying by column chromatography. The invention also provides a method for preparing p68 pertactin antigen by dissolving pertactin inclusion bodies in urea and optionally purifying by column chromatography.

In another embodiment, the composition is formulated for parenteral administration such that it further comprises a diluent or excipient for parenteral administration to a canine.

In another embodiment, the composition further comprises an antigen from a canine respiratory pathogen selected from the group consisting of: canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and Canine Influenza Virus (CIV). In more specific embodiments, the composition comprises at least two, three, or four antigens from a canine respiratory pathogen.

In another embodiment, the immunogenic composition described herein does not comprise a non-respiratory antigen. Accordingly, one embodiment of the present invention provides a composition as described herein, provided that it does not contain a non-respiratory antigen. The non-respiratory antigens do not cause respiratory disease in the subject. Non-limiting examples of such non-respiratory antigens include rabies virus, canine parvovirus, canine gastroenteritis virus, Leptospira spp (Leptospira species), and Borrelia burgdorferi (Borrelia burgdorferi).

In another embodiment, the antigen is from canine respiratory coronavirus (CRCoV). In another embodiment, the antigen is from Canine Influenza Virus (CIV). In another embodiment, the antigen is from canine parainfluenza virus (CPIV). In another embodiment, the antigen is from canine adenovirus-2 (CAV-2). In another embodiment, the antigen is from canine parainfluenza virus (CPIV) and canine adenovirus-2 (CAV-2).

In another embodiment, the composition comprises antigens from canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and Canine Influenza Virus (CIV).

In another embodiment, the composition induces an immune response to at least one of bordetella bronchiseptica and canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and Canine Influenza Virus (CIV).

Another embodiment of the invention provides an immunogenic composition comprising canine respiratory coronavirus (CRCoV), bordetella bronchiseptica preparation, and Canine Influenza Virus (CIV). More specifically, the compositions comprise an isolated pertactin antigen.

Another embodiment of the invention provides a method or use of the immunogenic composition of any of the embodiments described above for treating or preventing an infection from a canine respiratory pathogen in a canine. In another embodiment, the composition prevents infection from the canine respiratory pathogen in the dog for a period of 6 months or more. In another embodiment, the composition prevents infection from the canine respiratory pathogen in the dog for a period of 1 year.

In another embodiment, the canine respiratory pathogen is bordetella bronchiseptica. In another embodiment, the canine respiratory pathogen further comprises at least one, two, three, or four of the following: canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and Canine Influenza Virus (CIV).

Another embodiment of the present invention provides a method or use of the immunogenic composition of any of the above embodiments for treating or preventing canine infectious respiratory disease syndrome (CIRDC), wherein the composition treats or prevents infections from a plurality of canine respiratory pathogens. Another embodiment provides a use or method of parenteral administration of an immunogenic composition as described herein.

Another embodiment provides a pharmaceutical preparation comprising an immunogenic composition for use in treating or preventing infection in a canine from a canine respiratory pathogen.

These and other embodiments, features and advantages of the present invention will be apparent from the detailed description and appended claims set forth below. It is to be understood that the above and below described embodiments may be combined into a single embodiment.

Detailed Description

The following definitions apply to the present disclosure. Which supersedes any contradictory definitions contained in each individual reference incorporated by reference herein. The undefined terms have the meaning commonly used by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include both plural and plural forms, and plural forms shall include the singular.

"about" or "approximately" when used in connection with a measurable numerical variable refers to the value indicated for that variable, as well as all values of that variable that are within experimental error of the indicated value (e.g., within the 95% confidence interval of the mean), or all values of that variable that are within ten percent of the indicated value, whichever is greater. If "about" is used to refer to time intervals of weeks, "about 3 weeks" refers to 17-25 days, and "about 2 to about 4 weeks" refers to 10-40 days.

As used herein, "adjuvant" refers to any substance that acts as a non-specific stimulator of an immune response. See below for further description of adjuvants.

As used herein, the term "animal" includes any animal susceptible to bordetella bronchiseptica and/or canine respiratory disease syndrome, including domestic and wild-type mammals. Preferably, the animal as described herein refers to a dog or a human.

As used herein, an "antibody" is any polypeptide that protects an antigen-binding site, regardless of source, method of preparation, or other characteristics. It refers to the absence of a globin molecule or fragment thereof that specifically binds to an antigen as a result of an immune response to the antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains with "constant" and "variable" regions, and are classified based on the composition of the constant regions (e.g., IgA, IgD, IgE, IgG, and IgM). For given purposeAn antibody whose antigen is "specific" means that the variable region of the antibody specifically recognizes and binds to the specific antigen. As used herein, the indicated terms include, but are not limited to: polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, humanized antibodies, tetrameric antibodies, tetravalent antibodies, multispecific antibodies, single chain antibodies, domain-specific antibodies, single domain antibodies, domain-deleted antibodies (domain-deleted antibodies), fusion proteins, ScFc fusion proteins, single chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, mutated antibodies, and CDR-grafted antibodies. The antibody may be an intact immunoglobulin derived from a natural source or from a recombinant source, or may be an immunoreactive protein of an intact immunoglobulin. An "antibody" may be converted to an antigen binding protein, including but not limited to antibody fragments including but not limited to: fab, F (ab')₂Fab' fragments, Fv fragments, single chain Fv (scfv) fragments, Fd fragments, dAb fragments, diabodies, CDR3 peptides, constrained FR3-CDR3-FR4 peptides, nanobodies, diabodies, Small Modular Immunopharmaceuticals (SMIPs), and minibodies (minibodies), as well as any of the foregoing fragments and chemically or genetically manipulated ligands (counterpart), and other antibody fragments that retain antigen binding function. Typically, such fragments will comprise an antigen binding domain. As will be appreciated by those skilled in the art, any such fan can be engineered (e.g., "germlined") in order to reduce its immunogenicity, increase its affinity, alter its specificity or achieve other purposes.

As used herein, "antigen" or "immunogen" refers to a molecule containing one or more epitopes (linear epitopes, conformational epitopes, or both) that will induce an immune response specific for that antigen upon exposure to a subject. An epitope is a specific site of an antigen that binds to a T-cell receptor or a specific antibody and typically comprises from about 3 amino acid residues to about 20 amino acid residues. As used herein, the term antigen refers to subunit antigens-antigens that are separated and isolated from the intact organism with which the antigen is associated in nature-as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microorganisms. As used herein, the term also refers to antibodies, such as anti-idiotypic antibodies or fragments thereof, as well as to synthetic peptidomimetic positions capable of mimicking an antigen or antigenic determinant (epitope). The term antigen, as used herein, also refers to oligonucleotides or polynucleotides that express an antigen or antigenic determinant in vivo, such as in DNA immunization applications.

As used herein, "antigenicity" refers to the ability of a protein or polypeptide to immunospecifically bind to an antibody raised against the protein or polypeptide.

The term "Bordetella bronchiseptica (Bordetella bronchiatica or b. bronchiatica)" means: live attenuated bacteria of bordetella bronchiseptica, killed whole cell extracts (bacterins) of bordetella bronchiseptica or cell bacterial extracts of bordetella bronchiseptica.

"buffer" refers to a chemical system that prevents a change in the concentration of another chemical. The proton donor and acceptor system acts as a buffer, which prevents significant changes in hydrogen ion concentration (pH). Other examples of buffers include solutions containing weak acids and their salts (conjugate bases) or weak bases and their salts (conjugate acids).

As used herein, "dog" includes what is commonly referred to as a dog, but also includes other members of the canine family.

As used herein, the term "cell line" or "host cell" refers to a prokaryotic or eukaryotic cell in which a virus can replicate or be maintained.

As used herein, the term "culture" refers to a population of cells or microorganisms that grow in the absence of other species or types.

"dose" refers to a vaccine or immunogenic composition administered to a subject. By "first dose" or "priming dose" is meant the dose of such a composition administered on day 0. The "second dose" or "third dose" or "annual dose" refers to the amount of such composition administered after the first dose, which may be, but need not be, the same vaccine or immunogenic composition as the first dose.

An "epitope" is a specific site of an antigen that binds to a T-cell receptor or a specific antibody and typically comprises from about 3 amino acid residues to about 20 amino acid residues.

As used herein, "excipient" refers to the non-reactive carrier component of a vaccine or immunogenic composition, which is not an antigen. Preferred excipients are those known in the art for parenteral injection.

"fragment" refers to a truncated portion of a protein or gene. "functional fragment" and "biologically active fragment" refer to a fragment that retains the biological properties of the intact protein or gene.

"homology" or "percent homology" refers to the percentage of nucleotide or amino acid residues in a candidate sequence that are identical or similar to residues in a comparison sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology, and also considering any conservative substitutions as part of the sequence homology.

"homologs" or "species homologs" include genes found in two or more species that share substantial polynucleotide sequence homology and share the same or similar biological function and/or properties. Preferably, polynucleotide sequences representing species homologues will hybridise under moderately stringent conditions, as described by the examples herein, and have the same or similar biological activity and/or properties. In another aspect, polynucleotides representing species homologs will share greater than about 60% sequence homology, greater than about 70% sequence homology, greater than about 80% sequence homology, greater than about 90% sequence homology, greater than about 95% sequence homology, greater than about 96% sequence homology, greater than about 97% sequence homology, greater than about 98% sequence homology, or greater than about 99% sequence homology.

"identity" or "percent identity" refers to the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to residues in a compared sequence, after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, but without regard to any conservative substitutions as part of the sequence identity.

As used herein, an "immune response" in a subject refers to a humoral immune response, a cellular immune response, or a humoral and cellular immune response developed against an antigen. By "humoral immune response" is meant an immune response that is at least partially mediated by antibodies. A "cellular immune response" is an immune response mediated by T-lymphocytes or other leukocytes or both, and it includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, leukocytes or both. The immune response can be determined using standard immunoassays and neutralization assays known in the art.

As used herein, "immunogenic" refers to the ability of a protein or polypeptide to elicit an immune response specific to the bacteria or virus causing the disease.

An "immunogenic composition" is a preparation containing an immunogen, which includes, for example, a protein, peptide, whole cell, inactivated, subunit or attenuated virus, or polysaccharide, or a combination thereof, that is administered to stimulate the humoral and cellular immune response of a recipient to one or more antigens present in the immunogenic composition. "immunization" is the process of administering an immunogenic composition and stimulating an immune or immunogenic response to an antigen in a host. Preferred hosts are mammals, such as dogs. Preferably, the immunogenic composition is a vaccine.

As used herein. An "immunoprotective amount" is an amount of antigen effective to induce an immunogenic response in a recipient that is sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. Either humoral immunity or cell-mediated immunity or both can be induced. The immunogenic response of the animal to the composition can be evaluated, for example, indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms following challenge with the wild-type strain. For example, the protective immunity conferred by a composition or vaccine can be assessed by measuring the reduction in efflux (shed) of the attacking organism, the reduction in clinical signs such as mortality, morbidity, temperature, and overall physical health, and performance of the subject. The immune response may include, without limitation, induction of cellular and/or humoral immunity. The amount of the therapeutically effective composition or vaccine may vary and can be determined by a veterinarian depending on the particular organism used, or the condition of the animal being treated or vaccinated.

As used herein, "intranasal" administration refers to the introduction of a substance, such as a vaccine or other composition, into the body of a subject through or via the nose, and includes the delivery of the substance primarily through the nasal mucosa.

As used herein, the term "isolated" refers to a substance in a substantially pure form (e.g., greater than about 95% purity); or a substance that is purified or enriched in some way from its natural environment. Reference to "isolated pertactin" means that the pertactin protein is removed from its natural environment, such as from the host animal/dog, is located in a seed growth medium, or is purified from a whole cell bordetella bronchiseptica preparation, and which can then be added back to the composition containing the bordetella bronchiseptica extract (i.e., adding a pertactin isolate). As used herein, the term "isolated" includes the immunogen in a solution with other substances/diluents/excipients/adjuvants/proteins.

"agent" refers to any substance used to prevent, cure or improve a medical condition or to prevent some physiological condition or event.

As used herein, "monoclonal antibody" refers to an antibody produced by a single hybridoma cell line, all directed to one epitope on a particular antigen. The antigen used to prepare the monoclonal antibody may be provided as an isolated protein of the pathogen or as a whole pathogen. A "hybridoma" is a clonal cell line consisting of hybrid cells formed by the fusion of myeloma cells and specific antibody-producing cells. Generally, monoclonal cells are of mouse origin. However, monoclonal antibodies also refer to a clonal population of antibodies produced against a particular epitope of an antigen by phage display technology or equivalent to phage display methods or hybrid cells of non-mouse origin.

As used herein, "oral" or "oral" administration refers to the introduction of a substance, such as a vaccine or other composition, into the body of a subject by or via the oral cavity, and includes swallowing or delivery (e.g., sublingual or buccal absorption) or both, through the oral mucosa. Intratracheal administration is also a mode of oral or peroral administration.

As used herein, "oronasal" administration refers to the introduction of a substance, such as a composition or vaccine, into a subject via or via the nose and oral cavity, e.g., by placing one or more drops into the nose, as occurs. Oronasal administration includes delivery procedures associated with oral and nasal administration.

As used herein, "parenteral administration" refers to the introduction of a substance, such as a composition or vaccine, into a subject by or via a route that does not include the digestive tract. Parenteral administration includes subcutaneous, intramuscular, intraarterial, and intravenous administration. For the purposes of this disclosure, parenteral administration does not include routes of administration that primarily involve delivery of substances via mucosal tissues within the oral cavity, nose, organs, and lungs.

As used herein, the term "pathogen" or "pathogenic microorganism" refers to a microorganism, e.g., CPIV, CAV-2, CRCoV, mycoplasma canis, CIV or bordetella bronchiseptica, capable of inducing or causing a disease, disorder or abnormal state (preferably a respiratory disease, such as CIRDC) in its host animal.

As used herein, "pertactin" refers to an outer membrane protein of bordetella. Preferably. Pertactin is derived from bordetella bronchiseptica, more preferably from "p 68", and is encoded by the gene prnA. Pertactin can be isolated from bordetella bronchiseptica in its native form, or it can be recombinantly produced. Sequences and examples of pertactin are provided in U.S. patent No. 7,736,658, the contents of which are hereby incorporated by reference. Pertactin antigen as used herein includes lipidated forms of proteins.

"pharmaceutically acceptable" refers to a substance that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject, but that does not have undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for its intended use.

As used herein, "polyclonal antibodies" refers to a mixed population of antibodies directed against a particular pathogen or antigen. Generally, the population contains multiple antibody groups, each directed to a particular epitope of the pathogen or antigen. To produce polyclonal antibodies, whole pathogens or isolated antigens are introduced by inoculation into or infection of a host, which induces the host to produce antibodies against the pathogen or antigen.

As used herein, the term "polynucleotide" refers to an organic polymer molecule consisting of nucleotide monomers covalently bound in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides having different functions.

As used herein, the term "polypeptide" refers to an organic polymer molecule consisting of two or more amino acids bound in a chain.

As used herein, "preventing infection" refers to preventing or inhibiting the replication of the bacteria or viruses that cause the disease, so as to inhibit the spread of the bacteria or viruses, thereby preventing the bacteria or viruses from building themselves in their hosts, or alleviating the symptoms of the disease caused by infection. Treatment is considered therapeutic if there is a reduction in bacterial or viral load.

"protective", "protected", "protective immunity" as used herein with respect to a vaccine or other composition means that the vaccine or composition prevents or reduces the symptoms of disease caused by the organism from which the antigen used in the vaccine or composition is derived. The terms "protect", "protected", and the like, also refer to the vaccine or composition being useful for "treating" a disease or one or more symptoms of the disease already present in a subject.

As used herein, "respiratory" administration refers to the introduction of a substance of a vaccine or other composition into a subject by or via inhalation of a nebulized (aerosolized) substance. In respiratory applications, the primary delivery mechanism involves absorption of the aerosolized substance through the mucosa in the trachea, bronchi and lungs, and thus is different from intranasal or oral administration.

The term "specific binding (specific binding, specific bindings)" or the like is definedFor two or more molecules to form a complex that is measurable and selective under physiological or assay conditions. An antibody or other inhibitor is said to "specifically bind" to a protein if, under appropriately selected conditions, such binding is not substantially inhibited, while non-specific binding is inhibited. Specific binding is characterized by high affinity and selectivity for compounds or proteins. Non-specific binding typically has low affinity. Binding in IgG antibodies is generally characterized by a true about 10^-7M or higher affinity, such as about 10^-8M is or greater, or at least about 10^-9M is or greater, or at least about 10^-10Or higher, or at least about 10^-11M is or greater, or at least about 10^-12M or higher. As used herein, the term also applies, for example, to situations where an antigenic domain is specific for a particular epitope that is not carried by many antigens, in which case antibodies carrying the antigenic domain do not normally bind to other antigens.

As used herein, a "specific immunogenic fragment" refers to a sequence protein that is recognized only by an antibody or T cell specific for that sequence.

As used herein, "subject" refers to any animal having an immune system, including animals such as dogs.

As used herein, "substantially identical" refers to a degree of sequence identity of at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

As used herein, "subunit vaccine" and "subunit composition" refer to a class of vaccines or compositions that includes antigens derived from or homologous to (but not all antigens) a pathogen of interest, such as a virus, bacterium, parasite, or fungus. Such compositions or vaccines are substantially free of intact pathogen cells or pathogenic particles, or lysates of such cells or particles. Thus, a subunit vaccine or subunit composition can be prepared from an at least partially pure or substantially pure immunogenic polypeptide from a pathogen or analog thereof. Methods of obtaining the antigen or antigens in the subunit vaccine or subunit composition include standard purification techniques, recombinant production, or chemical synthesis. "subunit vaccine" or "subunit composition" thus refers to a vaccine or composition consisting of a defined antigenic component or components of a virus, bacterium or other immunogen. Preferably, the subunit component of the invention is recombinantly produced, and most preferably, it is pertactin (p 68).

“TCID₅₀"means" tissue culture infectious dose "and is defined as the viral dilution required to infect 50% of a given batch of inoculated cell culture. Various methods may be used to calculate the TCID₅₀Including the Spearman-Karber method, which is used throughout the specification. See b.w.mahy for a description of the Spearman-Karber method&H.O.Kangro,Virology Methods Manual 25-46(1996)。

As used herein, "therapeutic agent" refers to any molecule, mixture, virus, or therapeutic agent, preferably a virus attenuated or killed, or subunit or compound, that is useful in the treatment of viral, bacterial, parasitic, or fungal infections, and diseases or conditions caused thereby.

As used herein, "therapeutically effective amount" refers to an amount of antigen or vaccine or composition that will induce an immune response in a subject (e.g., a dog) receiving the antigen or vaccine or composition, which is suitable for preventing or alleviating the signs or symptoms of disease caused by infection with a pathogen, such as a virus, bacterium, parasite, or fungus, including adverse health effects or complications thereof. Either humoral immunity or cell-mediated immunity, or both humoral immunity and cell-mediated immunity, is induced. The immunogenic response of an animal to an antigen, vaccine or composition can be assessed indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms following challenge with the wild type strain. The protective immunity conferred by the vaccine or composition can be assessed by measuring a reduction in efflux of the challenge organism and/or a reduction in clinical symptoms such as mortality, morbidity, temperature, and overall physical health, and performance of the subject. The amount of therapeutically effective vaccine or composition may vary and can be determined by one skilled in the art depending on the particular immunogen used, or the condition of the subject.

As used herein, "treating" or treating "refers to reversing, alleviating, inhibiting the progression of, or preventing a disorder, condition, or disease to which such terms apply, or preventing one or more symptoms of the disorder, condition, or disease.

As used herein, "treatment" refers to the act of "treating" as defined above.

As used herein, "vaccine" or "vaccine composition" refers to an immunogenic composition selected from a virus or bacterium that is either modified active, attenuated or killed, or a subunit vaccine, or any combination of the foregoing. Administration of the vaccine to a subject generates an immune response. The vaccine can be introduced directly into the subject by known routes of administration, including parenteral, oral, etc. The term refers to a composition that prevents or reduces infection or one or more signs or symptoms of infection. Protection of vaccine compositions against pathogens is typically achieved by inducing an immune response in a subject. In general, the elimination or reduction of the incidence, signs or symptoms of infection or the accelerated elimination of the microorganism from the infected subject is indicative of the protective effect of the vaccine composition. The vaccine compositions of the present invention provide protection against infection by canine respiratory disease pathogens.

As used herein, "veterinarily acceptable" refers to materials which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a veterinary subject but which are not unduly toxic, irritating, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. As used herein, "veterinarily acceptable carrier" refers to a carrier medium that does not interfere with the efficacy of the biological activity of the active ingredient and is non-toxic to the veterinary subject to which it is administered.

Antigens, immunogenic compositions and vaccines

The present disclosure is based on the following unexpected findings: inclusion of isolated pertactin antigens in combination with bordetella bronchiseptica preparations resulted in substantially improved efficacy and safety. That is, the composition prevents the onset of bronchitis without producing undesirable side effects when administered parenterally to dogs prior to bacterial challenge.

The invention also provides immunogenic compositions and vaccines comprising one or more viruses and bacteria or subunits suitable for administration to a canine for treating CIRDC. A canine respiratory coronavirus (CRCoV) encompassed by the present invention can be characterized as a coronavirus that is present in the respiratory tract of a canine suffering from an infectious respiratory disease. CRCoV is most closely related to the following in the history of animal and plant evolution: bovine coronavirus (BCoV), human coronavirus (HCoV) strain OC43, and Hemagglutinating Encephalomyelitis Virus (HEV); enterocanine coronavirus (CCoV) is only distantly related to CRCoV. Representative examples of CRCoV suitable for use in the present invention include the strain identified as CRCoV strain 4182 (Erles et al, Virus Res.,124:78-87,2007).

The influenza virus antigen encompassed by the present invention may be any identified influenza virus strain from an avian or a mammal, including but not limited to: influenza viruses having the subtype H3 hemagglutinin and the subtype N8 neuraminidase or H3N8 subtype, which are more often referred to as H3N8 viruses. Influenza may be of any mammalian or avian origin, including but not limited to porcine, equine or canine origin. In one embodiment, a canine influenza antigen is used. In one embodiment, an equine influenza antigen is used. In one embodiment, strains with subtype glycoprotein designated H3 or N8 are used. In one embodiment, strains having the subtypes H3 and N8 glycoprotein are used.

Influenza antigens encompassed by the present invention can be isolated from domestic and wild dogs, horses, pigs and poultry. The animals selected for sample collection should exhibit acute and/or subacute clinical symptoms, which may include mild to severe respiratory symptoms and fever. Animals may also exhibit symptoms of anorexia and lethargy. Methods for virus isolation are well known to those skilled in the art and include: inoculation of mammalian or avian cell cultures, inoculation of embryonated eggs with nasal or laryngeal mucosal samples from clinical specimens, collection by swabbing the nasal passages or throat, or by collection of tissues such as the spleen, lungs, tonsils and liver and lung lavage. Cytopathic effects of the virus can be observed in cell culture. Allantoic fluid or cell lysates can be tested for their ability to accumulate human, chicken, turkey, or guinea pig erythrocytes, which is putative evidence of the presence of influenza virus.

Representative examples of Canine Influenza Virus (CIV) strains suitable for use in the present invention include the strain identified as A/canine/Iowa/9A 1/B5/08/D12, deposited at the American Type Culture Collection (ATCC) at 29.6.2006 with PTA-7694, 10801University Boulevard, Manassas, VA 20110-. Typical strains of CIV antigens are commercial vaccine CIV strains,

CIV (Pfizer). The invention also includes a vaccine comprising the strain identified as equine influenza strain A/horse/2/Miami/1/63. This strain was deposited with the ATCC under the number VR 317. Other examples of influenza viruses useful in the present invention are A/canine/Iowa/13628/2005, A/equine/Kentucky/1998, A/equine/Kentucky/15/2002, A/equine/Ohio/1/2003, A/equine/Kentucky/1/1994, A/equine/Massachusetts/213/2003, A/equine/Wisconsin/2003, A/equine/NewYork/1999, and A/equine/Newmarket/A2/1993. Other preferred CIV strains and/or isolates include those disclosed in U.S. patent nos. 7,959,929 (particularly the strains and HA sequences identified therein as Jacksonville/2005, Miami/2005, FL/242/03, and Florida/43/04), 7,384,642, 7,572,620, and 7,468,187, the contents of which (including all sequences, particularly the HA sequences and strains) are hereby incorporated by reference as if fully set forth herein. In addition, CIV strains suitable for use herein include Colorado CIV isolate numbered ADW41784 from Barrell et al, J.Vet.Intern.Med.,24(6),1524-1527 (2010).

The canine parainfluenza virus (CPIV) included in the present invention may be characterized as one of the viruses known to be the causative agent associated with kennel cough. Representative strains of CPIV antigens are attenuated CPI strains of commercial vaccines,

plus 5 (Pfizer). Another representative strain of CPIV antigen is the strain named "NL-CPI-5 "(National vector Service Laboratory, Ames, IA).

The canine adenovirus type 2(CAV-2) signature encompassed by the present invention may be one of the viruses known to be pathogens associated with kennel cough. Representative strains of CAV-2 antigens are attenuated CAV-2 strains of commercial vaccines,

plus 5 (Pfizer). A representative strain of CAV-2 antigen is an attenuated CAV-2 strain designated the "Manhattan" strain (National vector Service Laboratory, Ames, IA).

Mycoplasma canis (M.cynos) encompassed by the present invention is described in Chalker et al, Microbiology,150: 3491-. Immunogenic compositions against Mycoplasma canis are described in US2007/0098739, which is incorporated herein by reference.

The bordetella bronchiseptica component included in the present invention can be characterized as a bacterial pathogen associated with kennel cough. The immunogenic compositions and vaccines encompassed by the invention may be one or more of the following: live attenuated bordetella bronchiseptica, bordetella bronchiseptica bacterin or bacterial extract. In addition, the composition preferably further comprises an isolated subunit antigen of bordetella bronchiseptica.

In one embodiment, the bordetella bronchiseptica is prepared as whole cell sonicate (whole cell sonicate) purified by column chromatography, as provided in patent application No. FR2571618 filed 10/12/1984. Another representative example of Bordetella bronchiseptica is the bacterial extract Bronchicine^TMCae (pfizer), prepared from antigenic material extracted from bordetella bronchiseptica cells. Another example of Bordetella bronchiseptica is

Presented live attenuated bronchial sepsis strain B-C2 and/or from

Naramune^TM、

Univac and/or Kennel-Jec^TMThe live bronchial sepsis strain of (1).

In addition, the subunits are also preferably present in combination with (i.e., supplemented with) the bordetella bronchiseptica component. A representative example of such a subunit is an isolated pertactin antigen, preferably bordetella bronchiseptica p68 antigen, characterised by a recombinant bordetella bronchiseptica p68 antigen, which is recognized by the p 68-specific monoclonal antibody Bord2-7 (described in US 7,736,658, incorporated herein by reference), and in a preferred embodiment, has an amino acid sequence as described in US 7,736,658 or has homology thereto.

The recombinant p68 pertactin antigen is preferably prepared in a suitable form such that the native-like structure is preserved or restored during processing. Accordingly, one aspect of the invention provides recombinant p68 pertactin that is substantially free (less than about 80%, 90%, 95%, or even 99%) of aggregates. In another embodiment, recombinant p68 pertactin is dissolved with urea, preferably about a 0.1M, 0.5M, 1M, 2M, 3M or 6M urea solution. Thereafter, the p68 antigen can be purified, such as by column chromatography. One such dissolution method is described in Surender et al, J.bioscience and Bioengineering, v.99(4), pgs 303-.

Pertactin antigen as used herein includes lipidated forms. Examples of the preparation of lipidated proteins are provided in Erdie et al, Infection and Immunity, (1993) v.61(1) p.81-90, which is incorporated by reference. The methods disclosed therein can be used to prepare post-translationally modified pertactin proteins containing additional lipid moieties.

In addition, in another embodiment, the immunogenic composition comprises bordetella bronchiseptica and the isolated Bsp22 antigen. In another embodiment, the immunogenic composition comprises bordetella bronchiseptica, an isolated pertactin antigen, and further comprises an isolated Bsp22 antigen. Bsp22 antigen can be prepared as provided in Medhekar et al, Molecular Microbiology (2009)71(2), 492-504. Preferably, the isolated Bsp22 antigen is present in combination with a bordetella bronchiseptica extract and an isolated pertactin antigen (specifically recombinant p68) (i.e., including an isolated Bsp22 antigen in addition to the bordetella bronchiseptica extract and the isolated pertactin antigen). "Bsp 22" also includes lipidated forms of the antigen. Methods for the preparation of lipidated proteins are provided in Erdie et al, Infection and Immunity, (1993) v.61(1) p.81-90, which is incorporated by reference. The methods disclosed therein can be used to prepare a post-translationally modified Bsp22 protein containing an additional lipid moiety.

Viruses encompassed by the present invention can be propagated in cells, cytology and host cells. The cell, cytology or host cell may be, for example, but not limited to, mammalian cells and non-mammalian cells, including insect and plant cells. Viral cells, cytology and host cells in which the viruses encompassed by the present invention may be propagated are readily known and available to those skilled in the art.

The bacteria encompassed by the present invention can be cultured and propagated using a variety of media known to those skilled in the art, including broth (liquid) and agar (solid; semi-solid) media. Some bacteria may also be cultured and propagated in mammalian cells or non-mammalian cells.

Viruses and bacteria encompassed by the present invention may be attenuated or inactivated prior to use in an immunogenic composition or vaccine. Attenuation and inactivation methods are well known to those skilled in the art. Attenuation methods include, but are not limited to, serial passage in cell culture on suitable cell lines (viruses and some bacteria), serial passage in broth culture (bacteria), ultraviolet irradiation (viruses and bacteria), and chemical mutagenesis (viruses and bacteria). Methods of viral or bacterial inactivation include, but are not limited to, treatment with formalin, beta-propiolactone (BPL), or divinyl imine (BEI), or other methods known to those skilled in the art.

Inactivation by formalin may be performed by mixing the microorganism-containing suspension with 37% formaldehyde to form a final concentration of 0.5% formaldehyde. The microorganism-formaldehyde mixture was mixed by continuous stirring at room temperature for about 24 hours. The inactivated microbial mixture is then tested by measuring growth on a suitable cell line or broth culture.

For some antigens, inactivation by BEI may be performed by mixing a suspension containing the microorganism of the invention with 0.1M BEI (2-bromo-ethylamine in 0.175N NaOH) to a final BEI concentration of 1 mM. For the other antigens, the final BEI concentration was 2 mM. The person skilled in the art knows the appropriate concentration to be used. The virus-BEI mixture was mixed by stirring continuously at room temperature for about 48 hours and then adding 1.0M sodium thiosulfate to a final concentration of 0.1 mM. Mixing was continued for an additional 2 hours. The mixture containing the inactivated microorganisms is tested for residual live virus by measuring growth on a suitable cell line or broth culture.

The immunogenic compositions and vaccines encompassed by the present invention may include one or more veterinarily acceptable carriers. As used herein, "veterinarily acceptable carrier" includes any solvent, dispersion medium, coating, adjuvant, stabilizer, diluent, preservative, antibacterial and antifungal agent, isotonic agent, adsorption delaying agent, and the like. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, and lactose, as well as other isotonic agents known to those skilled in the art. Stabilizers include albumin and other stabilizers known to those skilled in the art. Preservatives include thimerosal as well as other preservatives known to those skilled in the art.

Adjuvants may be metabolizable, meaning that the adjuvant consists of components that are capable of being metabolized by the target organism, such as vegetable oil-based adjuvants. The metabolizable adjuvant may be a metabolizable oil. Metabolizable oils are fats and oils that are commonly found in plants and animals, and which are generally composed primarily of a mixture of triacylglycerols, also known as triglycerides or neutral fats. These non-polar water-insoluble substances are fatty acid triesters of glycerol. Triglycerides differ according to the identity and arrangement of their three fatty acid residues or side chains.

The adjuvant may also be non-metabolizable, meaning that the adjuvant is comprised of components that are not metabolized by the body of the animal subject to which the emulsion is administered. Non-metabolizable oils suitable for use in the compositions of the present invention include alkanes, alkenes, alkynes, as well as their corresponding acids and alcohols, their ethers and esters, and mixtures thereof. Preferably, the individual compounds of the oil are light hydrocarbon mixtures, i.e. such components have 6 to 30 carbon atoms. The oil may be synthetically prepared or purified from petroleum products. Preferred non-metabolizable oils for use in the compositions described herein include, for example, mineral oil, paraffin oil, and naphthenes. As used herein, the term "mineral oil" refers to non-metabolizable adjuvant oil that is a liquid hydrocarbon mixture derived from petrolatum via distillation techniques. As used herein, the terms "liquid paraffin", and "white mineral oil" are synonymous. As used herein, the term is also intended to include "light mineral oils", i.e., oils similarly obtained by petrolatum distillation, but which have a slightly lower specific gravity than white mineral oil. Mineral oils are available from various commercial sources, for example, j.t. baker (phillips burg, PA), USB Corporation (Cleveland, OH). Light mineral oils are commercially available under the name

Adjuvants include, but are not limited to, Emulsigen adjuvant systems (MVP Laboratories; Ralston, NE), RIBI adjuvant systems (Ribi Inc.; Hamilton, MT), alum, aluminum hydroxide gels, oil-in-water emulsions, water-in-oil emulsions (such as, for example, Freund's complete and incomplete additions), block polymers (CytRx; Atlanta, GA), SAF-M (Chiron; Emeryville, CA), and combinations thereof,

Adjuvants, saponins, Quil a, QS-21(Cambridge Biotech inc.; Cambridge, MA), GPI-0100(galenic Pharmaceuticals, inc.; Birmingham, AL) or other saponin fractions, monophosphoryl lipid a, avridine lipid-ammonia adjuvant, heat labile enterotoxin from e.coli (recombinant or other forms), cholera toxin, muramyl dipeptide, squalene/pluronic margarineSegmented copolymers/surfactants (SP-oil), sulfolipidyl beta-cyclodextrin (SL-CD), immunomodulator-containing liposomes (e.g., CpG or poly I: C), Muramyl Dipeptide (MDP), iscomatrix (Quil A/lecithin), CpG/DEAE-dextran/mineral oil (TXO), CpG, triterpenes (e.g., Quil A or another purified or partially purified saponin preparation), sterols (e.g., cholesterol), immunomodulators (e.g., dioctadecyldimethylammonium bromide-DDA), polymers (e.g., polyacrylic acids, such as polyacrylic acid

) And Th2 stimulants (e.g., glycolipids, such as Bay

) And combinations thereof, as well as many of their adjuvants known to those skilled in the art.

Other non-limiting examples of various combinations that can be used include triterpenoids plus sterols (e.g., Quil a/cholesterol, also known as QAC), triterpenoids plus sterols, immunomodulators and polymers (e.g., Quil a/cholesterol/DDA @)

Also known as QCDC) and triterpenoids plus sterols, immunomodulators, polymers and Th2 agonists (e.g., Quil a/cholesterol/DDA @)

And Bay

Also known as QCDCR).

The amounts and concentrations of adjuvants and additives used in the context of the present invention can be readily determined by one skilled in the art. In one embodiment, the invention includes immunogenic compositions and vaccines comprising about 20 μ g to about 2000 μ g of adjuvant. In another embodiment, the adjuvant is included in an amount of about 100 μ g to about 1500 μ g, or about 250 μ g to about 1000 μ g, or about 350 μ g to about 750 μ g. In another embodiment, the adjuvant is included in an amount of about 500 μ g/2ml of immunogenic composition or vaccine.

The immunogenic compositions and vaccines also include antibiotics. Such antibiotics include, but are not limited to, those selected from the classes of aminoglycosides, carbapenems, cephalosporins, glycopeptides, macrolides, penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines. In one embodiment, the invention includes immunogenic compositions and vaccines comprising from about 1 μ g/ml to about 60 μ g/ml antibiotic. In another embodiment, the immunogenic compositions and vaccines comprise from about 5 μ g/ml to about 55 μ g/ml antibiotic, or from about 10 μ g/ml to about 50 μ g/ml antibiotic, or from about 15 μ g/ml to about 45 μ g/ml antibiotic, or from about 20 μ g/ml to about 40 μ g/ml antibiotic, or from about 25 μ g/ml to about 35 μ g/ml antibiotic. In yet another embodiment, the immunogenic compositions and vaccines comprise less than about 30 μ g/ml of antibiotic.

Immunogenic compositions and vaccines encompassed by the present invention may include one or more polynucleotide molecules encoding viral or bacterial or viral or bacterial proteins. DNA or RNA molecules may be used in the immunogenic composition or vaccine. The DNA or RNA molecule can be administered in the absence of other substances, or it can be administered without a substance that promotes cellular uptake (e.g., liposomes or cationic liposomes). The total polynucleotides in the immunogenic composition or vaccine is typically between about 0.1 μ g/ml to about 5.0 mg/ml. In another embodiment, the total polynucleotides in the immunogenic composition or vaccine is from about 1 μ g/ml to about 4.0mg/ml, or from about 10 μ g/ml to about 3.0mg/ml, or from about 100 μ g/ml to about 2.0 mg/ml. Vaccines and vaccination procedures utilizing nucleic acids (DNA or mRNA) are well described in the prior art, for example, U.S. Pat. No. 5,703,055, U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,589,466, all of which are incorporated herein by reference.

In addition to the viruses or bacteria described above, the immunogenic compositions and vaccines encompassed by the present invention may include other additional antigens. The antigen may be an inactivated whole or partial preparation of the microorganism, or an antigenic molecule obtained by genetic process techniques or chemical synthesis. Other antigens suitable for use in accordance with the present invention include, but are not limited to, those derived from: pathogenic viruses such as canine distemper virus, canine herpes virus, canine influenza virus, rabies virus; pathogenic bacteria such as Leptospira bratislava (Leptospira bratislava), Leptospira canicola (Leptospira canicola), Leptospira grippotyphosa (Leptospira grippotypha), Leptospira icterohaemorrhagiae (Leptospira icterohaemorrhagiae), Leptospira pomona (Leptospira pomona), Leptospira hardjoovis (Leptospira hardjoovis), Porphyromonas (Porphyromonas spp.), bacteroides (Bacteriodes spp.), Borrelia (Borrelia spp.), Streptococcus (Streptococcus spp.), including Streptococcus subspecies zooepidemicus (Streptococcus spp.), and Mycoplasma (Mycoplasma spp.), Mycoplasma pneumoniae (Mycoplasma spp.). The antigen may also be derived from a pathogenic fungus, such as candida, from a protozoan, such as cryptosporidium, neospora, toxoplasma, eimeria, babesia, giardia, leishmania, or from a parasite, such as cestodes, flavomyiasis, echinococcus and paragonimus.

Form, dosage and route of administration

The immunogenic compositions and vaccines encompassed by the present invention can be administered to animals to induce an effective immune response against CIRDC. Accordingly, the present invention provides methods of stimulating an effective immune response by administering to an animal a therapeutically effective amount of an immunogenic composition or vaccine described herein.

The immunogenic compositions and vaccines described herein can be administered to an animal in order to vaccinate the animal against CIRDC vaccine. The immunogenic compositions and vaccines can be administered to an animal to prevent or treat CIRDC in the animal. Thus, described herein are methods of vaccinating an animal against CIRDC, and methods of preventing or treating CIRDC, comprising administering to the animal a therapeutically effective amount of an immunogenic composition or vaccine described herein.

The immunogenic compositions and vaccines encompassed by the present invention may be prepared in a variety of forms depending on the route of administration. For example, the immunogenic compositions and vaccines can be prepared as sterile aqueous solutions or dispersions suitable for injectable use, or in lyophilized form using freeze-drying techniques. Lyophilized immunogenic compositions and vaccines are typically maintained at about 4 ℃ and can be reconstituted in a stable solution (e.g., saline or HEPES) with or without adjuvant. The immunogenic compositions and vaccines can be prepared in the form of a suspension or emulsion.

The immunogenic compositions and vaccines of the present invention comprise a therapeutically effective amount of one or more of the above microorganisms. The purified virus and/or bacteria may be used directly in an immunogenic composition or vaccine, or may be further attenuated or inactivated. Typically, the immunogenic composition or vaccine comprises about 1X 10²To about 1X 10¹²Viral or bacterial particles, or about 1X 10³To about 1X 10¹¹Granules, or about 1X 10⁴To about 1X 10¹⁰Granules, or about 1X 10⁵To about 1X 10⁹Granules, or about 1X 10⁶To about 1X 10⁸And (3) granules. The precise amount of microorganism effective to enhance the protective effect in an immunogenic composition or vaccine can be determined by one skilled in the art.

The immunogenic compositions and vaccines typically comprise a veterinarily acceptable carrier in a volume of about 0.5ml to about 5 ml. In another embodiment, the volume of the carrier is from about 1ml to about 4, or from about 2ml to about 3 ml. In another embodiment, the volume of the carrier is about 1ml, or about 2ml, or about 5 ml. The veterinarily acceptable carrier suitable for use in the immunogenic compositions and vaccines can be any of those described above.

One skilled in the art can readily determine whether a virus or bacterium requires attenuation or inactivation prior to administration. In another embodiment of the invention, the virus or bacterium can be administered directly to the animal without additional attenuation. The amount of microorganisms that are therapeutically effective may vary depending on the particular microorganism used, the condition of the animal, and/or the extent of infection, and can be determined by one skilled in the art.

In accordance with the methods of the invention, a single dose may be administered to an animal, or alternatively, two or more vaccinations may be performed at intervals of about two weeks to about ten weeks. A booster regimen may be required and the dosage regimen may be adjusted to provide optimal immunization. The optimal administration regimen can be readily determined by one skilled in the art.

The immunogenic compositions and vaccines can be administered directly into the bloodstream, intramuscularly, in the gut, or subcutaneously. Suitable methods for parenteral administration include intravenous, intraarterial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle (including micro-needle) syringes and needle-free syringes.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates, proteins and buffers (preferably to a pH of from about 3 to about 9, from about 4 to about 8, or from about 5 to about 7.5, or from about 6 to about 7.5, or from about 7 to about 7.5), but in some applications may be more suitably formulated as a sterile non-aqueous solution or in dry form, or used in combination with a suitable vehicle (sterile, pyrogen-free water or saline).

Preparation of parenteral formulations under sterile conditions by, for example, lyophilization, can be readily accomplished using standard pharmaceutical techniques known to those skilled in the art.

The solubility of the materials used in the preparation of the parenteral solution can be increased using suitable formulation techniques known to those skilled in the art, such as the introduction of solubility enhancing agents, including buffers, salts, surfactants, liposomes, cyclodextrins, and the like.

Compositions for parenteral administration may be formulated for immediate release or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release. Thus, the immunogenic compositions and vaccines can be formulated as solid, semi-solid, or thixotropic liquids for administration as an implant depot, providing for the modified release of the immunogenic compositions and vaccines.

Other means of administration of the immunogenic composition or vaccine include through a micro-needle or needle-free (e.g., Powderject)^TM,Bioject^TMEtc.) injection.

In the case of subcutaneous or intramuscular injection, an isotonic formulation is preferred. In general, additives for isotonicity may include chlorocyclo, dextrose, mannitol, sorbitol, and lactose. In particular instances, isotonic solutions, such as phosphate buffered saline, are employed. The formulation may also include stabilizers such as oxu gel and albumin. In some embodiments, a vasoconstrictor is added to the formulation. The pharmaceutical formulations according to the invention are prepared sterile and pyrogen-free. However, for any canine vaccine, polypeptide (antigen) subunit immunogenic compositions and vaccines, recombinant viral vector vaccines and DNA vaccines, those skilled in the art are familiar with the formulation of pharmaceutically acceptable carriers as those pharmaceutical carriers approved by the united states department of agriculture or equivalent foreign government agencies such as canada or mexico, or in any of the european national published regulations. Thus, a pharmaceutically acceptable carrier for commercial production of an immunogenic composition or vaccine is one that has been approved or will be approved by a suitable governmental agency in the united states or foreign countries. The immunogenic compositions and vaccines can also be mixed with pharmaceutically acceptable adjuvants. In certain formulations of the immunogenic compositions and vaccines, the immunogenic compositions or vaccines are combined with other canine immunogenic compositions or vaccines to produce a multivalent product that protects dogs from a variety of diseases caused by canine pathogens.

Detection and diagnostic methods

The extent and nature of the immune response induced in an animal can be assessed by using various techniques. For example, serum can be collected from the vaccinated animal and tested for the presence or absence of antibodies specific for the immunogen. Detection of corresponding cytotoxic T-lymphocytes (CTLs) in lymph tissue may be achieved by assays such as T cell proliferation, which indicates that a cellular immune response is induced. Related art is fully described in the prior art.

Reagent kit

Since it may be desirable to administer the immunogenic composition or vaccine in combination with other compositions or mixtures, for example for the purpose of treating a particular disease or condition, it is within the scope of the present invention that the immunogenic composition or vaccine may suitably be included in, or incorporated into, a kit form suitable for administration or co-administration of the composition.

Thus, kits encompassed by the present invention may comprise one or more separate pharmaceutical compositions, at least one of which is an immunogenic composition or vaccine according to the present invention; and means for individually holding the compositions, such as a container, a divided bottle, or a divided foil package. Examples of such kits are syringes and needles and the like. The kits of the invention are particularly suitable for administration of different dosage forms, e.g. oral or parenteral dosage forms, for administration of separate compositions at different dosage intervals, or for titration of different compositions with respect to each other. To facilitate administration of the compositions encompassed by the present invention, the kit typically includes instructions for administration.

The invention further encompasses a kit for protecting one or more reagents for detecting an infected animal. The kit may include reagents for assaying a sample for the presence of an intact microorganism, polypeptide, epitope or polynucleotide sequence. The presence of viral, bacterial, polypeptide or polynucleotide sequences can be determined using antibody, PCR, hybridization and other detection methods known to those skilled in the art.

Another kit encompassed by the present invention may provide reagents for detecting antibodies against a particular epitope. Such reagents are used to analyze a sample for the presence of antibodies and are readily known and available to those skilled in the art. The presence of antibodies can be determined using standard detection methods known to those skilled in the art.

In certain embodiments, the kit can include a set of printed instructions or a label indicating that the kit is to be used to detect an infected animal.

Antibodies

The antibody may be monoclonal, polyclonal or recombinant. Antibodies can be prepared against immunogens or portions thereof. For example, synthetic peptides based on the amino acid sequence of the immunogen or recombinantly produced synthetic peptides by cloning techniques, or native gene products and/or portions thereof, can be isolated and usedAn immunogen. The antibody may be produced using an immunogen by standard antibody production techniques well known to those skilled in the art. Antibody fragments can also be prepared from antibodies by methods known to those skilled in the art, and include Fab, F (ab')₂And Fv fragments.

In producing antibodies, screening for the desired antibody can be accomplished by standard methods in immunity known in the art. In general, ELISAs and immunoblotting are preferred types of immunoassays. Both assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assay. The antibody may be bound to a solid support substrate, conjugated to a detectable moiety, or bound and conjugated as is well known in the art. The binding of antibodies to solids is also well known in the art. Detectable moieties contemplated for use in the present invention may include, but are not limited to, fluorescent, metallic, enzymatic, and radioactive labels such as biotin, gold, ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, gamma-glucosidase, gamma-peroxidase, gamma-glucosidase, gamma-peroxidase, gamma-glucosidase, or a combination thereof,¹⁴C and iodination.

The invention is further illustrated by the following examples, but is in no way limited to them.

Examples

Example 1: immunogenicity of bordetella bronchiseptica bacterial extract subunit vaccines

32 beagle dogs with low bordetella bronchiseptica (microagglutinating antibody (MAT) titers <16) were involved in the study at about 8 weeks of age. Puppies were randomly divided into two treatment groups (T1 and T2), 16 per group. One puppy was removed from T2 prior to inoculation, due to an inguinal hernia.

Dogs were vaccinated subcutaneously with the appropriate vaccine on days 0 and 21 according to the study design shown in table 1. The vaccine was administered to each dog in the right shoulder area at the first vaccination and in the left shoulder area at the second vaccination.

Dogs from all treatment groups were challenged intranasally with bordetella bronchiseptica on day 42 by nebulization in the Plexiglas chamber for 30 minutes. Four dogs were challenged at one time, 2 from T1, 2 from T2, one group being excluded, which had only 3 animals, 2 from T1, and 1 from T2, since one animal had been previously removed from the study.

The primary efficacy variable is cough. All other variables (nasal discharge, ocular mucus, sneezing, depression, retching, respiratory sounds) are considered supportive secondary variables. Signs of respiratory illness (including coughing) were observed clinically twice a day ("observation periods"; am and pm), with each period being about 30 minutes per room, from day 42 until day 70 (end of study). Post-challenge (post-challenge) was calculated for each animal, regardless of whether the animal coughed during two consecutive observations. The frequency distribution of coughing/non-coughing for each animal during two consecutive observations was calculated. The coughing/non-coughing during two consecutive observations was analyzed with the Cohran-Armitage test, adjusted for the room, since it was not possible to analyze the data with a generalized linear mixture model. The frequency distribution (+/-) of nasal bacterial isolation was calculated for each treatment and time point. It was also determined whether each animal had bacteria isolated after challenge (bacterio-isolated post-challenge).

For each treatment group, the frequency distribution of whether the animals had bordetella bronchiseptica isolated after challenge was calculated and, if possible, analyzed using a generalized linear mixture model. The duration of nasal outflow (nasal shedding post-challenge) after challenge was calculated for each animal and analyzed using a generalized linear mixture model. Antibody titers were logarithmically transformed and duplicate measurements were analyzed using a generalized linear mixture model.

As a result: no pain or fever (>39.5 ℃) was reported in any dog after vaccination. Some dogs in both treatment groups were reported to have had scratches at the time of inoculation. Only two dogs in T2 reported small-size injection swelling (2 x 1cm) during the 3-6 hour period after the first vaccination observation. The data indicate that vaccination is safe and well tolerated by dogs.

All saline controls developed coughing, indicating adequate challenge. The Least Squares (LS) mean of the duration of cough in the control group was 20.3. In contrast, in vaccination, the vaccine significantly reduced the duration of cough to an average LS of 5.2, and a p-value of <.0001. In addition, other clinical signs of the respiratory tract, such as retching and respiratory sounds, were reduced in the vaccination when compared to controls.

Post challenge bacterial isolation data showed that the test vaccine significantly reduced the duration of nasal outflow of challenge organisms in the vaccinated group (LS mean 20.5) compared to the saline control (LS mean 25.3) and a p-value of 0.0061. This data indicates that the vaccine is effective in reducing the level of infection as evidenced by a reduction in the duration of shed (shedding) in vaccination.

The antibody responses generated after vaccination in the T2 group (days 21 and 42) were similar to T1, probably due to the nature of the agglutinated antibody assay, and did not lack antigenicity. Compared to the saline control (LS mean 13), at day 70, a strong previous response was observed to be induced in most post challenge vaccinations (LS mean 426), indicating that the immunization was effective.

Overall, the data from this study clearly show that bordetella bronchiseptica bacterial extract (bacterial extract) subunit vaccines exhibit efficacy by reducing the duration of cough and nasal effluence.

Example 2: evaluation of efficacy and safety of injectable Bordetella bronchiseptica vaccine

50 beagle dogs of about 8 weeks of age were involved in the study. Dogs were randomly divided into 5 treatment groups of 10 dogs each. All animals were in good health and did not receive any bordetella vaccination. The prescreened serum samples were negative for bordetella bronchiseptica and had titers in the microaggregation test (MAT) before the first inoculation of < 16. All animals were determined to be free of bordetella bronchiseptica by a bacterial nasal swab isolation assay prior to first inoculation (day 0).

Animals were vaccinated with the appropriate vaccine on days 0 and 21 according to the study design shown in table 2. The vaccines of the T01, T02, T03 and T04 groups were administered subcutaneously in the right shoulder region to each dog for the first vaccination, and in the left shoulder region for the second vaccination. Group T05 received a single intranasal vaccination with a commercially available intranasal vaccine on day 0. The group was finally vaccinated to prevent live vaccine spread in the dwelling and the dogs in the group were housed separately in different rooms to prevent exposure to other groups.

Dogs from all treatment groups were challenged intranasally with bordetella bronchiseptica on day 42 by nebulization in the Plexiglas chamber for a total of 30 minutes. Five dogs from the same pen (one for each treatment group) were challenged at one time.

The primary efficacy variable is cough. Bacteria were isolated as secondary variables. Two times a day ("observation periods"; am and pm) clinical observations of signs of respiratory illness included coughing), with about 30 minutes per room, from day 42 through day 62, and once on day 63 (am) (end of study). The percentage of observation period for cough was transformed with a square root arcsine transform prior to cough analysis. The percentage of change during cough observation and the number of days of cough were analyzed using a generalized linear mixture model. For each animal, the percentage of the observed period during which the animal coughed after challenge and the number of days that coughed occurred after challenge were calculated.

As a result: slight to moderate swelling was observed at the injection sites of the dogs of treatment groups T02 and T04 (data not shown). The severity of the response and the number of dogs involved increased after the second vaccination. Mild injection site reactions were observed in several dogs in treatment group T03. No measurable injection swelling was observed in saline group T01. No clinical fever was observed (>39.5 ℃), and only one dog in the T02 group was on day 4 after inoculation. No pain was reported in any of the vaccinated dogs.

Bordetella bronchiseptica challenge vaccination induced cough (53.3%, cough 14.7 days) in all unvaccinated controls, indicating that challenge was appropriate for evaluation of the test vaccine. The results of the resulting T05 group, which received a commercial intranasal vaccine (positive control), showed an observed cough of 4.6% up to 2 days after challenge, indicating that the level of challenge was optimal and not overwhelming.

Three test formulations were evaluated in this study. T02 had 16.1% observed cough up to day 6 post challenge, T03 had 34.7% observed cough up to day 10.5 post challenge, and T04 had 34.5% observed cough up to day 10.7 post challenge. Thus, all vaccinated groups showed a reduced cough score compared to the unvaccinated control. T02 group inoculated with bordetella bronchiseptica bacterial extract supplemented with pertactin and adjuvant QCDC showed a statistically significant reduction in cough score criteria. Thus, the efficacy of the formulation tested in T02 was found to be effective.

Example 3Safety and efficacy of Bordetella bronchiseptica-containing vaccines in dogs

Fifty (50) dogs were selected for the study and divided into 5 treatment groups. Whether the animals were suitable for the study was determined based on the-4 th day of physical examination.

Blood samples (approximately 8mL) were collected from all animals in SST tubes on days-2, 21 and 28 of the study prior to each vaccination for serology. Serum samples collected on day-2 were used to confirm that the animals did not contain bordetella bronchiseptica. Nasal swabs were collected on day 0 prior to inoculation and tested for the presence of bordetella bronchiseptica. Tympanic membrane temperatures were collected starting on day-4 to establish baseline prior to inoculation.

Dogs were vaccinated with the appropriate vaccines on days 0, 21 and 28 according to the study design shown in table 3. The vaccine was administered subcutaneously to each dog in the right shoulder area at the first vaccination and in the left shoulder area at the second vaccination.

Post-inoculation injection site responses were observed for all animals on days 0, 21 and 28 of inoculation. The animals were observed daily for injection response after inoculation from days 1-7 and days 22-35. Tympanic membrane temperatures were collected on days 0-7 and days 21-35.

Blood samples (approximately 6mL) were collected on the day before challenge, i.e. day 55, for serology. Tympanic membrane temperatures were collected on days 54, 55 and 56 prior to challenge. Nasal swabs were collected on the day prior to challenge, i.e., day 55, and tested for the presence of bordetella bronchiseptica. On days 54 and 55, twice daily (morning and afternoon), and on the morning of day 56, approximately 30 minutes during each period, the animals were observed for clinical symptoms of respiratory illness in order to establish baseline values.

Preparation of 10 by attacking strains of Bordetella bronchiseptica⁹CFU/4 mL/target challenge dose in dogs. Dogs from all treatment groups were challenged intranasally with bordetella bronchiseptica on day 56 by nebulizing challenged pens in Plexiglas chambers for a total of 30 minutes. Five dogs from the same pen (one for each treatment group) were challenged at one time.

Tympanic membrane temperature was recorded once daily from day 56-77 after challenge. Signs of respiratory illness (including coughing) were observed clinically twice a day ("observation periods"; am and pm), approximately 30 minutes per room, from day 56 until day 76, and once on day 77. Briefly, cough, nasal discharge, sneezing, ocular mucus, retching and depression were observed using the following scoring systems: none (0), mild (1), moderate (2) and severe (3). Nasal swabs were collected on days 59, 62, 66, 69, 74, 76 and 77 and assayed for efflux of challenge organisms.

Blood samples (approximately 6mL) were collected on day 77 for serology. Nasal swabs were collected using swabs and delivery medium for isolation of bordetella bronchiseptica.

Aggregated antibodies to bordetella bronchiseptica were measured by the Microaggregation Assay (MAT). From days 0, 28, 55 and 77, serum samples from treatment groups T04 and T05 were titrated, CRCoV antibodies were determined by seroneutralization and IFA, and CIV was determined by HAI. Isolation of bordetella bronchiseptica from nasal swabs was performed according to standard procedures. Each sample was qualitatively tested for the presence or absence of bacteria.

ResultsFifty (50) healthy beagle beagles of about 8 weeks of age on day 0, were isolated by nasal swab culture and confirmed to be free of bordetella bronchiseptica organisms. Evaluation of serum samples for bordetella bronchiseptica agglutination antibodies by MAT confirmed that all beagle dogs were susceptible to infection on day-2 with MAT titers of 8.

All experimental vaccines evaluated in this study yielded a slight to no injection swelling after the first vaccination. For most vaccinations, injection swelling was limited to day 0 of the study. Slight to no injection swelling was also reported after the second vaccination. At the time of this occurrence, injection site swelling subsided one to three days after the second vaccination. Scratch was reported mainly in the 5-pathway combination group (T04). No clinical fever was reported after vaccination. No injection swelling was reported in the saline group. The data confirm the safety of the vaccine.

Colony counts performed before and after challenge inoculation confirmed that, on average, 1.45x10⁸CFU bordetella/dogs were aerosolized in the room. Challenge vaccination induced cough in all saline control dogs (T02), with an average observed percentage of cough of 43.5% and 12.2 days of cough. Similar to the saline control, treatment group T05 vaccinated with only 4-pathway virus (CRCoV/CIV/CPIV/CAV2) without bordetella antigen developed coughing with an average observed percentage of cough of 43.4% and a number of coughing days of 12.2 days. These findings indicate that challenge was appropriate and consistent for evaluation of the test vaccine.

Dogs in the bordetella vaccine vaccinated treated group T01 were significantly protected from challenge (cough 3.6 days, p <0.0001) when compared to the control group (cough 12.2 days). The same vaccine also significantly protected dogs in T03 when administered at 3 weeks intervals (cough 5.8 days, p ═ 0.0004). The reduction in cough score was not significantly different in the two groups (T01 versus T03) (p-value 0.1883), indicating that the level of protection was similar for the vaccines administered at 3-or 4-week intervals.

Dogs in T04 receiving the unadjuvanted 5-way combination vaccine were significantly (p 0.0016) protected against bordetella challenge (cough 6.6 days) when compared to the control group (cough 12.2 days) and to T05 receiving the combination of 4-way virus (CRCoV/CIV/CPIV/CAV2) (cough 12.1 days, p 0.0019), demonstrating the efficacy of the bordetella moiety in the adjuvanted combination vaccine.

Serological evaluation of the viral fractions in the 5-way combination vaccine is possible for only two fractions. CIV and CRCoV, where dogs were seronegative as demonstrated on study day-2. On study day 56, the CIV HAI response numbers in the 4-way vaccine group (T04) were similar to the 5-way vaccine group (T05), suggesting that there was no interference of the bordetella moiety with CIV antigens. At day 56 of the study, the CRCoV SN response in the 4-pathway vaccine group (T04) was numerically higher than the 5-pathway vaccine group (T05), suggesting that bordetella may have interference with the CRCoV moiety. However, these findings are not conclusive, as these vaccines are unadjuvanted, and the formulations are not optimized, and no CRCoV challenge is performed to test efficacy.

Monovalent bordetella vaccines have proven to be safe and effective. The efficacy of a monovalent vaccine was demonstrated when the vaccine was administered at 21 or 28 day intervals. The bordetella fraction also showed efficacy when administered as a 5-way adjuvant-free combination vaccine.

Example 4.Multivalent serological Studies

Fifty dogs, which were about 8 weeks old and in good health, were pre-screened for bordetella bronchiseptica by the microaggregation test (MAT) and canine respiratory coronavirus (CRCoV) by indirect fluorescent antibody assay (IFA). Serum Neutralization (SN) was also used to assess antibody levels. On day 0, all dogs were negative for antibodies against Bordetella bronchiseptica as determined by MAT (< 16), and all dogs were negative for antibodies against CRCoV as determined by IFA (< 40). All dogs also did not contain bordetella bronchiseptica and CRCoV prior to first inoculation (day 0), as determined by nasal swab isolation assay.

Dogs were divided into 5 treatment groups of 8 dogs each and vaccinated according to the study design shown in table 4. The vaccine was administered to each dog in the right shoulder area at the first vaccination and in the left shoulder area at the second vaccination.

After the second vaccination, the T04 and T05 groups were removed from the study due to complications. The dogs in the remaining groups (T01, T02 and T03) were observed daily for post-vaccination reactions and body temperature (tympanic membrane) was monitored after each vaccination for 7 consecutive days. Blood samples were collected from dogs on days 0, 21, 42 and 56 to measure antibody responses.

Serum samples from days 0, 21, 42 and 56 were tested for agglutinating antibodies against bordetella bronchiseptica by the MAT assay. Serum samples from the same day were titrated to determine CRCoV antibodies by serum neutralization, CIV by HAI, and CAV-2 and CPI antibodies by serum neutralization. Geometric mean antibody titers were obtained for each treatment group.

As a result:

after the second dose, the test vaccines in T02 and T03 groups induced antibody responses in all (100%) vaccinated dogs, indicating auto-immunization against viral antigens. In most breeder dogs, the antibody response increased after the second vaccination, indicating that the second vaccination had a boosting effect. It is important to note that in the presence of multiple viral and bacterial (bordetella bronchiseptica) antigens, antibody responses were achieved between the viral parts, indicating the absence of immune interference. MAT serology is not associated with defense against bordetella but can be a valuable screening tool for introduction into appropriate research animals. In general, the efficacy of viral antigens is predicted in a 5-way multivalent vaccine based on the immune response in the immunized dogs.

Example 5.Duration of immunization study

A. Unit price:

the objective of this study was to demonstrate the duration of immunity against viral bordetella bronchiseptica challenge in dogs of 8 weeks of age in a bordetella bronchiseptica extract-subunit vaccine.

All animals were in good health and did not receive any bordetella vaccination. Prior to the first vaccination, the animals had low (16) or no antibody titer against Bordetella bronchiseptica, as determined by the microaggregation test (MAT). All animals also did not contain bordetella bronchiseptica prior to the first inoculation, as determined by the bacterial nasal swab isolation assay.

The dogs were divided into 2 treatment groups; one group received a placebo vaccine and the other group received a bordetella bronchiseptica extract supplemented with recombinant antigen. The antigen is pertactin, Bsp22, or both. Animals were vaccinated twice, approximately 3-4 weeks apart. After each vaccination, injection site reactions were observed.

Dogs of all treatment groups were challenged by nebulization with bordetella bronchiseptica approximately 6-12 months after inoculation. Challenge dose and purity of challenge inoculum were determined before and after challenge. Clinical observations were performed before and after challenge.

Nasal swabs were collected for isolation of bordetella bronchiseptica. Blood was collected from each animal and used for serum. Agglutinating antibodies to bordetella bronchiseptica were determined by MAT. Serum samples were titrated for pertactin-specific IgG antibody responses using ELISA. Isolation of bordetella bronchiseptica from nasal swabs was performed according to standard procedures.

Cough, bordetella isolation (post challenge) and post vaccination serological responses are criteria used to judge vaccine efficacy in the study.

B. Polyvalent:

the purpose of this study was to demonstrate the duration of immunity of multivalent respiratory combination vaccines in dogs. The vaccine contains the following antigen components: modified-live CAV-2, modified-live CPIV, inactivated CIV, inactivated CRCoV, and bordetella bronchiseptica extract supplemented with recombinant antigens that are pertactin, Bsp22, or both.

All animals were in good health and did not receive vaccination with any of the pathogens that the vaccine was intended to protect against. Dogs were divided into multiple treatment groups. Each group consisted of two treatment groups. The control group received a placebo vaccine and the vaccination group received the test vaccine. Animals were vaccinated twice, approximately 2-4 weeks apart. After each vaccination, injection site reactions were observed.

Approximately 6-12 months after vaccination, both treatment groups (vaccinated and control) were challenged with one of the pathogens that the vaccine was intended to protect against. Clinical observations were performed before and after challenge. During the post-challenge period, nasal swabs were collected for isolation of challenge pathogens. Blood from each animal was collected for serum acquisition for subsequent analytical analysis.

Clinical symptoms of respiratory disease, pathogen efflux after challenge and serological response are criteria used to judge vaccine efficacy.

Having thus described in detail embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof.

All references mentioned above are hereby incorporated by reference as if fully set forth herein.

Claims (9)

1. An immunogenic composition for use in a dog comprising bordetella bronchiseptica and an isolated bordetella pertussis adhesin antigen, wherein said isolated bordetella pertussis adhesin antigen is an isolated bordetella bronchiseptica p68 protein, wherein said p68 protein is present in an amount of 10 to 20 ug per dose.

2. The immunogenic composition of claim 1, wherein the composition further comprises an antigen from a canine respiratory pathogen selected from the group consisting of: canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and Mycoplasma canis (M. Cynos).

3. The immunogenic composition of claim 2, wherein the composition comprises at least two, three or four antigens from the canine respiratory pathogen.

4. The immunogenic composition of claim 2, wherein the composition comprises antigens from canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), and canine respiratory coronavirus (CRCoV).

5. Use of an immunogenic composition as claimed in any preceding claim in the manufacture of a medicament for use in preventing infection in a dog from a respiratory pathogen of said dog.

6. The use of claim 5, wherein the composition prevents infection by the canine respiratory pathogen in the dog for a period of 6 months or more.

7. The use of claim 5 or 6, wherein the canine respiratory pathogen is Bordetella bronchiseptica.

8. The use of claim 7, wherein the canine respiratory pathogen further comprises at least one, two or three of: canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), and canine respiratory coronavirus (CRCoV).

9. Use of the immunogenic composition of any one of claims 2-4 in the manufacture of a medicament for preventing canine infectious respiratory disease syndrome (CIRDC), wherein the medicament prevents infections from a plurality of the pathogens, wherein the canine infectious respiratory disease syndrome is caused by a pathogen selected from the group consisting of: bordetella bronchiseptica, canine parainfluenza virus (CPIV), canine adenovirus-2 (CAV-2), canine respiratory coronavirus (CRCoV), and mycoplasma canis (m.